Diethylnitrosamine causes pituitary damage, disturbs hormone levels, and reduces sexual dimorphism of certain liver functions in the rat.

The acute toxicity of diethylnitrosamine (DEN) to the liver has been well documented in the literature, but whether DEN also affects the endocrine parameters has been addressed in only a few studies. We thus investigated the effects of DEN on pituitary, serum hormone levels, and certain sex-differentiated liver enzymes in this study. Adult male Wister rats were intraperitoneally injected with DEN at a single dose of 200 mg/kg and were sacrificed at 1, 3, 7, and 35 days after injection; DEN-treated females were included as controls at days 7 and 35. Electron microscopic observation showed that during the first week after injection, all types of granular cells of the anterior pituitary in male animals exhibited cellular damage, including disrupted organelles and cellular structure, as well as pyknotic or lytic nuclei. Many undamaged secretory cells exhibited dilated endoplasmic reticula, hypertrophic Golgi complexes, and peripheral location of secretory granules, which usually are morphologic features of increased cellular activities. In male rats, the serum level of total testosterone decreased and the corticosterone increased 1 day after DEN treatment. The serum level of growth hormone (GH) decreased and the prolactin level increased on day 3. The hepatic expression of the male-specific cytochrome P450 2C11 (CYP2C11) decreased to 1-5% of the normal levels during the first week and was still 50% lower than the normal level on day 35, whereas the female-specific CYP2C12 expression increased only slightly. Activities of the male predominant 16alpha, 16beta, and 6beta hydroxylation of androstenedione by microsome decreased in an in vitro assay, whereas the non-sex-differentiated 7alpha hydroxylation and the female-predominant 5alpha reduction of androstenedione were unaffected. In female rats, decreased serum GH level was observed on day 7. The CYP2C12 expression in females was decreased to about 1% and 80% of the normal levels on day 7 and day 35, respectively, but the CYP2C11 expression was unchanged. These data suggest that in male rats, DEN treatment may cause pituitary damage, disturb serum hormone levels, and induce long-lasting reduction of sexual dimorphism in certain liver functions.

Inhibition of in vivo rat liver regeneration by 2-acetylaminofluorene affects the regulation of cell cycle-related proteins.

The effects of dietary 2-acetylaminofluorene (2-AAF) on cell cycle-related proteins was studied in regenerating livers from male Wistar rats. The levels of cyclins, cyclin dependent kinases (cdks), and related proteins were studied at different times during the first cell cycle after partial hepatectomy (PH). The frequency of proliferation cell nuclear antigen (PCNA)-positive nuclei, a marker of S phase progression, was almost zero during the first 27 hours after PH in the mitoinhibited 2-AAF-treated rats, while about 50% of the nuclei were labeled 24 hours after PH in control animals. Accordingly, Western blot tests showed markedly elevated PCNA protein levels from 18 hours to the end of S phase in untreated animals but no upregulation in response to 2-AAF. Compared with control animals, animals treated with 2-AAF showed increased levels of cdk 4 and cyclin D3 from 12 and 15 hours after PH, respectively, and altered cyclicity in cyclin D3 expression. No effects on cyclin E were observed, while the increase in cdk 2 levels in control animals during late G1/S (15-27 hours) was abolished by 2-AAF. p53 was induced by 2-AAF treatment during the same period, with a peak at 24 hours. The protein detected with p21 antibodies was highly expressed in unstimulated hepatocytes in control animals, and further increased by 2-AAF. The expression was sustained until 15 hours after PH in control rats while 2-AAF-treated animals lacked detectable protein during this period; however, a transient increase was observed at 21 hours. Thus, 2-AAF affects several parameters of cell cycle regulation of possible relevance for its inhibitory effects on hepatocyte proliferation in vivo.

Role of the pituitary in tumor promotion with ethinyl estradiol in rat liver.

Synthetic estrogens act as tumor promoters in rat liver. Because estrogen treatment markedly increases the secretion of pituitary prolactin, also shown to be a tumor promoter in rat liver, the possibility of a pituitary influence in estrogen promotion was investigated in Wistar rats. In diethylnitrosamine (DEN)-initiated hypophysectomized (hx) female rats, 24 weeks of ethinyl estradiol (EE) administration (500 microg/kg/d, intraperitoneally) did not increase the number of hepatocyte nodules and did not induce hepatocellular carcinoma (HCC) in a 2-year study. Very few placental forms of glutathione-S-transferase (GST-P)-positive foci were observed at the end of EE administration. Estrogen receptor (ER) messenger RNA (mRNA) levels in hx females were 20% of the levels in intact females. EE administration (range, 160-210 microg/kg/d, subcutaneous release pellets) to DEN-initiated intact males and females increased the number and size of hepatocyte foci. A significant increase in HCC frequency was observed in EE-treated females compared with females receiving sham-release pellets, and the latency period for HCC induction was decreased by EE in both males and females. Inhibition of prolactin (PRL) secretion by bromocriptine (Brc) (ParlodelLAR, slow intramuscular release vehicles) during EE treatment decreased the number of foci without affecting their size and markedly prolonged the latency period in both sexes. EE treatment also significantly increased the expression of c-myc, and c-jun, enhanced the levels of growth hormone receptor (GHr) mRNA in females and the levels of ER mRNA in males and "feminized" the expression of the GH-regulated genes cytochrome P450 (CYP), 2C11, CYP 2C12, and GHr in male liver. Brc administration decreased the mRNA levels of the female-predominant CYP 2C12 in EE-treated males but otherwise had no effects. In conclusion, a decreased promotive effect of EE was obtained by decreasing the PRL levels, indicating that estrogens exert at least part of their promotion effects indirectly, by increasing the levels of pituitary PRL.

Expression of the c-jun, jun-B, ets-2 and liver regeneration factor-1 (LRF-1) genes during promotion and progression of rat liver carcinogenesis in the resistant hepatocyte model.

During promotion in the RH-model, the mRNA expression of c-jun and LRF-1 was 2- to 8-fold elevated in both initiated and uninitiated rats receiving 2-AAF. The increase was more pronounced in male than in female rats, and GH treatment of male rats down-regulated the expression towards the level in females. The level in uninitiated 2-AAF-treated livers was as high as in isolated early nodules. jun-B also showed 3- to 8-fold increased expression, but without sex differences. An increased nuclear transcription of the LRF-1 and jun-B genes but not of c-jun was observed. During progression, LRF-1 and ets-2 showed a 2- to 3-fold higher expression in persistent nodules and hepatocellular carcinomas than in the corresponding surrounding liver tissues, whereas the expression of the jun genes was 3- to 4-fold increased both in lesions and in surrounding livers when compared to age-matched control rats. In conclusion, while the changes during promotion might not be connected with control of early focal growth, the increased levels of LRF-1 and ets-2 in advanced lesions might indicate that these genes could contribute to the growth advantage for persistent nodules during progression.

Role of protein kinase C in growth stimulation of primary mouse colonic epithelial cells.

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (160 nM) and the secondary bile acid, deoxycholic acid (50 microM) stimulated DNA synthesis in quiescent primary epithelial cells from the normal mouse colon as measured by autoradiographic analysis of [3H]thymidine incorporation. The purpose of this present study was to investigate the involvement of protein kinase C in the proliferative response of the normal colonic cells. The protein kinase C inhibitor, bisindolyl-maleimide GF 109203X, efficiently blocked the proliferative response of the cells to the phorbol ester and caused a dose-dependent decrease in the response to deoxycholic acid. While the phorbol ester-induced proliferation was unaffected by another inhibitor, H-7, the response of the cells to deoxycholic acid was blocked. Pretreatment of the cells with the phorbol ester (160 nM) for 24 h blocked the proliferative response to deoxycholic acid. Measurement of the intracellular distribution of protein kinase C activity showed a time-dependent and significant translocation of the enzyme activity from the soluble to the particulate cell fractions after exposure to 12-O-tetradecanoylphorbol-13-acetate. While exposure to the bile acid indicated a similar time-dependent translocation of the enzyme activity, the effect was not significant. The phorbol ester induced a time-dependent accumulation of c-fos mRNA and protein was measured by solution hybridization and immunocytochemistry, respectively. No effect of deoxycholic acid on c-fos expression could be observed in the present study. The data support a role for protein kinase C in the growth stimulating effect of physiological concentrations of deoxycholic acid on normal colonic epithelial cells. However, differences in the mechanisms underlying phorbol ester- and bile acid-induced proliferation are indicated.

Hormonal regulation of sex differentiated parameters in liver nodules from rats treated in the resistant hepatocyte model.

Sex differentiation of liver functions has been shown to be attenuated in preneoplastic rat liver nodules. The present study was performed to investigate whether nodules from male rats are to some extent withdrawn from the normal growth hormone (GH) regulation of these functions. Male and female Wistar rats were treated according to a modified resistant hepatocyte model (RH-model), with diethylnitrosamine initiation and promotion with intragastric administration of 2-acetylaminofluorene (2-AAF) combined with partial hepatectomy (PH). Eleven months post-initiation male rats were treated with either human (hGH) or bovine growth hormone (bGH) or ovine prolactin (oPRL) by continuous infusion for 1 week. The mRNA expression of a number of genes known to be sex differentiated in liver from adult control rats was compared in nodular and surrounding tissue from nodule-bearing male, female and hormone-treated male rats. The basal mRNA expression of the female-predominant cytochrome P4502C12 (CYP2C12) was increased and the male-predominant CYP2C11 was decreased in liver nodules from male rats compared with the surrounding liver. Expression of the prolactin receptor (PRL-r; female > male) and the steroid 5 alpha-reductase (female > male) genes was decreased in male nodules, whereas no difference was observed with respect to GH-receptor (GH-r; female > male) expression in nodules versus surrounding tissue. Early nodules obtained from males treated according to the original RH-model (dietary 2-AAF, 0.02%) and isolated 2 weeks after completion of the 2-AAF/PH treatment showed significantly lower GH-r mRNA levels than the total liver tissue. In hepatocellular carcinomas from hormonally unmanipulated males 11 months post-initiation the decrease in PRL-r expression was even more pronounced than in the nodules and a significant decrease in GH-r expression was seen. In female nodules the only significant difference with respect to the sex differentiated parameters was a lower 5 alpha-reductase expression than in the surrounding tissue. Continuous infusion of both hGH and bGH feminized the expression of all the sex differentiated genes in male tissues and eliminated the previously detected differences between nodules and surrounding tissue. oPRL also eliminated the differences between nodules and surrounding tissue in males and partly feminized the expression of both the 5 alpha-reductase and the PRL-r genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of the liver-enriched transcription factors C/EBP alpha, C/EBP beta, HNF-1, and HNF-4 in preneoplastic nodules and hepatocellular carcinoma in rat liver.

The expression patterns of the liver-enriched transcription factors CCAAT/enhancer-binding protein (C/EBP) alpha and beta and hepatocyte nuclear factor (HNF)-1 and HNF-4 were studied in liver nodules and hepatocellular carcinomas from male rats treated according to the resistant hepatocyte (RH) model. C/EBP alpha expression was lower at the transcriptional, mRNA, and protein levels in persistent nodules than in the respective surrounding livers. Expression was further decreased in the tumors. Transcriptional downregulation of C/EBP alpha gene expression was observed already in very early nodules, isolated 3 wk after partial hepatectomy in the RH model. However, no detectable changes were observed in preneoplastic nodules in the transcription or in steady-state mRNA levels of C/EBP beta, HNF-1, and HNF-4. A slight decrease in C/EBP beta protein and a more pronounced attenuation of HNF-1 and HNF-4 levels was observed in nodules, being 67%, 37%, and 46% of the levels in the corresponding surrounding livers, respectively. In conclusion, differential regulation of several transcription factors that are associated with the maintenance of the differentiated state of the hepatocytes was observed in preneoplastic and neoplastic liver lesions. This could have an impact on the regulation of a wide array of genes during liver carcinogenesis. Furthermore, the attenuation of C/EBP alpha expression, regarded as a negative growth regulator, could contribute to the proliferative advantage of nodules during liver carcinogenesis.

Growth hormone administration after treatment in the resistant hepatocyte model does not affect progression of rat liver carcinogenesis.

Male and female Wistar rats were treated according to a slightly modified resistant hepatocyte model, i.e. initiation with diethylnitrosamine and selection of initiated cells with 2-acetylaminofluorene and partial hepatectomy. Two weeks after selection, rats of each sex received daily subcutaneous injections of either recombinant human growth hormone (2.5 IU/kg) or saline for 6 weeks. No effects on growth of early enzyme-altered liver lesions were recorded. The long-term part of the experiment did not show any differences due to growth hormone treatment in terms of incidence or latency time for development of either malignant liver tumors or kidney tumors. Male rats developed liver tumors more frequently than the female rats whereas a higher incidence of kidney tumors was observed in the female rats. Several different malignancies at other sites were also recorded, with no differences between the groups with or without growth hormone treatment. In conclusion, no modifying effects of human growth hormone administration during the post selection phase of the resistant hepatocyte model could be demonstrated on either tumor promotion or tumor progression.

Increased expression of the female-predominant cytochrome P4502C12 in liver nodules from male Wistar rats.

Nodules and hepatomas from male and female rats treated according to the resistant hepatocyte (RH) model were analyzed with respect to expression of the male-predominant cytochrome P4502C11 (P450(16 alpha)) and the female-predominant cytochrome P4502C12 (P450(15 beta)) at the transcriptional and at the mRNA and protein levels. In male nodules isolated 8 and 11 to 12 months after initiation and in hepatomas, the expression of P450(16 alpha) mRNA was 3- to 11-fold lower than in surrounding liver, whereas a 2- to 8-fold higher expression of P450(15 beta) was observed compared with surrounding tissue. These alterations in P450 mRNA expression were reflected by similar changes at the protein level. Nuclear transcription of the cytochrome P450(16 alpha) gene was lower in male nodules than in surrounding liver whereas transcription of the P450(15 beta) gene was higher in the nodules. In nodules and hepatomas from female rats no significant differences in either mRNA expression or protein level of either P450(16 alpha) or P450(15 beta) were seen. The present study indicates that liver nodules are, to some extent, withdrawn from the normal endocrine regulation of rat liver function. Furthermore, the observed increase in a specific cytochrome P450 species (P450(15 beta)) in male liver nodules contradicts the previous suggestion of a general downregulation of this enzyme family as a characteristic of the nodular phenotype.

Role of growth hormone in the regulation of the c-myc gene during progression of sex-differentiated rat liver carcinogenesis in the resistant hepatocyte model.

In this study, we extended the previous observations of a growth hormone-regulated sex difference in hepatic c-myc expression in the resistant hepatocyte model during the selection/promotion phase, when sex differences in growth rate of enzyme-altered foci are first identified, to studies of the regulation of this gene during later stages of hepatocarcinogenesis. The expression of the c-myc gene was studied in preneoplastic nodules, hepatocellular carcinomas, and the corresponding surrounding livers of male and female Wistar rats treated according to the resistant hepatocyte model. In males, nodules isolated 8 and 11-12 mo after initiation and hepatocellular carcinomas exhibited a 2.5- to threefold higher c-myc expression than the surrounding liver. In females, no increase in c-myc expression was observed in nodules 8 mo after initiation, while nodules isolated after 11-14 mo and tumors showed a twofold and threefold increase, respectively, when compared with the surrounding tissue. Increased transcription of the c-myc gene was observed in nuclei from male nodules isolated 11 mo after initiation compared with nuclei from the surrounding liver. The difference in transcription between male nodules and surrounding tissue is similar for the first and second exon of the gene. Continuous infusion of growth hormone to nodule-bearing male rats 8 and 11 mo after initiation decreased c-myc expression in the surrounding tissue and downregulated the expression in 8-mo nodules to the level in the surrounding liver. No significant decrease in response to growth hormone treatment was seen in 11-mo nodules. In hypophysectomized nodule-bearing males, nodular c-myc remained upregulated. Taken together, the data showed that the sex difference in c-myc expression was maintained during a large part of the progression period. Furthermore, the loss of growth hormone regulation of the c-myc gene in advanced male nodules indicated an escape from normal regulatory mechanisms during progression. These findings might reflect a role for the c-myc gene in sex-differentiated rat liver carcinogenesis.

Hypothalamo-pituitary regulation of the c-myc gene in rat liver.

Expression of the c-myc gene was studied in the livers of male and female Wistar rats. Furthermore, the effects on hepatic c-myc expression of neonatal and adult castration, with or without testosterone supplementation, as well as of continuous administration of GH to intact males, were analysed. Expression of c-myc was low in 6-day-old animals of both sexes, reached a maximum at 35 days of age and declined to the level of adult animals at 70 days. In prepubertal animals, expression was higher in females, but was higher in males after the onset of puberty, the postpubertal female rat liver exhibiting 50-70% of the expression in males. Treatment of adult male rats with bovine GH in osmotic minipumps for 1 week reduced c-myc expression to the level of female rats. Castration, both neonatally and of adults, also feminized hepatic c-myc expression. Testosterone supplementation of the castrated animals increased the expression towards the level in sham-operated controls. These results indicate that the c-myc gene is regulated by the hypothalamo-pituitary-liver axis via the sex-differentiated pattern of GH secretion, in analogy with other sex-differentiated hepatic functions, such as metabolism of steroids and xenobiotics. Neuroendocrine regulation of a gene such as c-myc, which is involved in the control of cell proliferation and differentiation, represents another aspect of the complex influence of GH on various somatic functions.

Loss of sexual differentiation of metabolism of steroids and xenobiotics in nodular hepatic tissue from male and female Wistar rats treated according to the resistant hepatocyte model.

Male and female rats were treated according to the resistant hepatocyte model, i.e. initiation with diethylnitrosamine (DEN) and selection/promotion with 2-acetylaminofluorene (2-AAF) and partial hepatectomy (PH). Non-initiated controls were either treated with 2-AAF/PH or with PH only. Initiated male controls, where PH was performed, were also included. In livers obtained at PH marked effects of treatment with DEN and/or 2-AAF on several cytochrome P450-mediated microsomal reactions towards steroid and xenobiotic substrates were observed. Hepatic N,O-sulfation of N-hydroxy-2-acetylaminofluorene (N-OH-2-AAF) was decreased in rats of both sexes in response to 2-AAF treatment at the time of PH. Microsomes prepared from early male nodules, collected 39 days after initiation, exhibited levels of cytochrome P450 similar to that in liver from non-initiated male rats treated with 2-AAF/PH. The microsomal content of cytochrome P450 in nodules from both male and female rats, at 8 and 11 months after the start of the experiment respectively, was lower than that of the surrounding liver. No differences in the metabolism of 4-androstene-3,17-dione (androstenedione) were observed in early male nodules compared with 2-AAF/PH-treated controls. Eight months after initiation several sex differentiated hydroxylations of androstenedione (male greater than female) were lower in nodules than in surrounding and control liver from male rats. Female nodules obtained 11 months post-initiation exhibited a markedly lower capacity for 5 alpha-reduction of androstenedione (male greater than female) than the surrounding liver, whereas no significant differences were observed with respect to the different hydroxylation pathways. N,O-Sulfation of N-OH-2-AAF was the only reaction that was markedly decreased in preparations from early male nodules, compared with livers from non-initiated 2-AAF/PH-treated males. Also in late male nodules the sulfotransferase activity was lower than in the surrounding liver. At 11 months, N,O-sulfation in preparations from female rat liver did not reach the detection level. In conclusion, several enzyme activities are markedly less sex differentiated in nodular tissue from male and female rats than in surrounding tissue or in different kinds of control rat liver. These findings indicate that hepatocyte nodules are to some extent withdrawn from the normal endocrine regulation of rat liver.

Effects of growth hormone on the expression of c-myc and c-fos during early stages of sex-differentiated rat liver carcinogenesis in the resistant hepatocyte model.

The expression of the oncogenes c-myc and c-fos was studied in Wistar rats treated according to the resistant hepatocyte model. During early phases of promotion, i.e. the first 4 days after partial hepatectomy (PH) when the growth hormone (GH)-dependent sex difference in outgrowth of preneoplastic foci becomes manifest, c-myc and c-fos expression were compared in livers from males, females and GH-treated males. The expression of c-fos was almost doubled in males when compared to females 1 day after PH, a difference that gradually declined during the following days. In males receiving growth hormone in osmotic minipumps from 1 week after initiation the expression was at about the same level as that in females, c-myc expression was enhanced in males from the first day after PH and remained elevated, with a maximal 2.5-fold increase at day 2, while the expression in females and GH-treated males was practically unchanged. The increased c-myc expression in males and the effect of GH was still apparent 28 days after PH. Nuclear transcription assay showed a 2- to 3-fold higher transcription of the c-myc gene in untreated when compared with GH-treated males at the third day after PH. In conclusion, continuous GH infusion was shown to modulate the expression of c-myc and c-fos during the phase when sex-differentiated promotion is first observed. These findings might reflect a connection between the regulation of these genes and promotion of liver carcinogenesis. They might also be of possible importance for the understanding of the mechanism of hormone-related cancer.